Water and Buffers.

In the Beginning

• Quote by Carl Sagan: "It is far better to grasp the universe as it really is than to persist in delusion, however satisfying and reassuring."

Introduction: Basic Biology

• Diversity of living organisms is astounding.
  • Estimates: ~8.7 million eukaryotic species.
  • Bacteria: between $10^7$ and $10^9$ different species.
  • Archaea: expected to be very large.
• Organisms occupy diverse environmental niches.
  • Human gut, Antarctic, Amazon rainforest, acid mine waste.
• Tardigrades (water bears) withstand incredibly harsh conditions.
  • Few degrees above absolute zero to 300°F.
  • Vacuum of outer space.
  • Pressures greater than ocean depths.
• Dr. Ian Malcolm in Jurassic Park: “Life finds a way”.
• Common characteristics shared by all living things.
  • All living things are made up of cells.
  • Discovered by Robert Hooke in 1665.
  • Observed slices of cork under a microscope; saw tiny chambers named cells.
• All three domains of life (bacteria, archaea, eukaryotes) are made up of cells.
  • Some are single-celled, while others are multicellular.
• Biochemistry: the science that explains life at the molecular level.
  • The characteristics of cells influence the chemistry of life.

Cells

• All cells, regardless of type, have common features:
  • Plasma membrane: boundary separating the cell from surroundings.
  • Genome (DNA): encodes information for making proteins.
  • Ribosomes and tRNAs: machinery for protein synthesis.
  • DNA: repository of information copied and transmitted to next generation.
  • Need to obtain and use energy.
• Energy sources vary among organisms.
  • Phototrophs: obtain metabolic energy from light.
  • Chemotrophs: obtain energy from the oxidation of chemical fuels.
  • Autotrophs: capture energy from light or chemical sources (auto=self,
    troph=nourishing).
  • Heterotrophs: use organic compounds made by other organisms as their energy source.
    • Plants and photosynthetic organisms are autotrophs.
    • Animals are heterotrophs.
• Cells may be aerobic (use oxygen) or anaerobic (live without oxygen).
  • Obligate anaerobes: require an environment free of oxygen.
  • Facultative anaerobes: can live with or without oxygen.

Prokaryotic and Eukaryotic Cells

• Organisms divided into prokaryotes and eukaryotes.
  • Prokaryotes: lack a nucleus and other organelles.
  • Eukaryotes: have numerous internal, membrane-bounded compartments, including a
    nucleus.
• Prokaryotes:
  • Unicellular.
  • Generally smaller than eukaryotic cells (0.5 to 5 µm in diameter).
  • Typically have circular chromosomes.
  • May contain extra-chromosomal DNA elements (plasmids - usually circular).
  • DNA is not wrapped around histones but has associated proteins.
    • DNA-protein complexes create a structure called a nucleoid.
    • Nucleoid differs from the eukaryotic nucleus (not enclosed by a nuclear
      envelope).
    • Proteins associated with DNA differ among Archaea (resemble eukaryotic
      histones), Bacteria (different from both eukaryotic and archaeal).
• Prokaryotes divided into bacteria and archaea.
  • Both ancient and widespread.
  • Archaea once thought to be subgroup of bacteria; now a completely different
    group in their own domain.

Bacteria

• Like eukaryotic cells, bacterial cells have a plasma membrane surrounding them.
• They contain an exterior cell wall, comprised of an interlocked peptidoglycan network.
• Exterior surfaces have hair-like appendages called pili for adhering to other cells.
  • Pili play a role in bacterial conjugation (DNA transfer between bacterial cells).
• Bacterial cells may have flagella enabling them to move.
• Bacteria can communicate with members of their own species and other bacterial species using chemical signals (quorum sensing).
  • Quorum sensing helps bacteria assess conditions (e.g., population size).
  • Plays a role in the process of infection by bacterial pathogens.
  • Formation of biofilms (mats of cells) that:
    • adhere to each other.
    • protect the bacteria against environmental hazards/harmful agents.

Archaea

• First archaeans studied found in harsh environments (salt flats, hot springs).
  • Initially believed to live only in extreme environments and were described as
    extremophiles.
• Archaea found in every environment, moderate or extreme, including the human
  gut.
• Marine plankton contain huge numbers; possibly the most abundant organisms on
  earth.
• Unicellular, superficially resemble bacteria, but in some respects more similar
  to eukaryotes.
• Transcriptional machinery, promoter sequences and ribosomes are more like
  those of eukaryotes than of prokaryotes.
• Unique use of ether linkages to join lipids in plasma membranes to glycerol.
  • Ether linkages differ from ester linkages.
  • Lipids themselves are different (long isoprene-derived chains instead of fatty
    acids).
• Difference in membrane composition/structure: archaeal membranes are highly
  stable, advantageous in extreme conditions.
• Like bacteria, archaea also have a cell wall, but the cell walls do not contain peptidoglycans.
  • Some archaea have peptidoglycan-like molecules in their cell walls.
  • Others build cell walls entirely of glycoproteins and polysaccharides.

Eukaryotes

• Eukaryotic cells are found in both unicellular and multicellular organizational schemes.
  • Unicellular: yeast, many protists (Paramecium, Amoeba).
  • Multicellular: plants, animals, fungi.
• Eukaryotic cells are surrounded by a plasma membrane.
• Animal cells have no cell walls, whereas plant cells use:
  • cellulose.
  • hemicellulose.
  • pectins
• to build cell walls outside their plasma membranes.
• Fungal cells have unusual cell walls containing chitin (also in arthropod
  exoskeletons).
• Eukaryotic cells are typically much larger (typically 10-100 µm) and contain
  considerably more DNA than prokaryotic cells.
• The most distinctive feature: the presence of a variety of internal,
  membrane-bounded structures, called organelles.

Organelles

• Eukaryotic cells characterized by internal membrane-bounded compartments,
  or organelles.
• These compartments divide up the interior of the cell into discrete parts that
  have specialized functions.
• Organelles found in eukaryotic cells include:
  • Nucleus (houses DNA).
  • Mitochondria (electron transport system/oxidative phosphorylation for ATP
    synthesis).
  • Nucleolus (ribosome synthesis and assembly).
  • Endoplasmic reticulum (lipid metabolism and targeted protein synthesis and
    folding).
  • Golgi apparatus (protein modification and secretion).
  • Peroxisomes (oxidation of very long chain fatty acids).
  • Chloroplasts (plants - photosynthesis).
  • Plastids (synthesis and storage of compounds in plants).
  • Lysosomes (animals - hydrolytic enzymes).
  • Endosomes (contain endocytosed material).
  • Vacuoles.
• Multiple compartments permit reactions requiring specific conditions to be carried out in isolation from the rest of the cell.
  • Disulfide bond formation (possible in endoplasmic reticulum, but not cytosol).
• Allow reactants to be more concentrated (smaller volume of the organelle).

Chromosomes

• Eukaryotic DNA is divided into several linear bundles called chromosomes.
• Chromosomes contain the genomic DNA wrapped around cores of positively
  charged proteins called histones.
• Ends of linear eukaryotic chromosomes have telomeres (short sequences repeated thousands of times).
  • Telomeres prevent loss of information when linear chromosomes are replicated.
• Chromosomes surrounded by the nuclear envelope (double membrane structure).
• Enzymes required for replication and transcription of genetic information.
• Nuclear envelope regulates which proteins can enter the nucleus.
  • Important way to control gene expression.
• Mitochondria and chloroplasts have their own DNA, separate from nuclear DNA.
  • Small and circular; resembles a prokaryotic chromosome.
  • They also have their own ribosomes and tRNAs and can carry out their own
    protein synthesis.
  • These organelles are likely derived from prokaryotes that once lived as
    endosymbionts within ancient eukaryotic cells and eventually became
    integrated into their host cells.

Cytoskeleton

• Another interesting feature of eukaryotic cells is the presence of an internal
  skeleton-like structure called a cytoskeleton.
• The cytoskeleton is made up of a network of interlinking protein fibers belonging to three major classes:
  • Microtubules.
  • Microfilaments (actin filaments).
  • Intermediate filaments.
• All eukaryotic cells have microfilaments and microtubules, but plant cells may
  lack intermediate filaments.
• Some cytoskeletal elements are present in bacteria and archaea as well.
• Although the word “skeleton” may suggest a rigid and fixed structure, the cytoskeleton is dynamic.
  • Microfilaments and microtubules disassemble and rearrange themselves on an ongoing
    basis, as needed.
  • Intermediate filaments are also broken down and rebuilt, at specific times,
    such as during cell division.
• Three main classes of cytoskeletal elements distinguished by:
  • proteins that they are composed of.
  • way in which those proteins assemble into structures seen in the cell.
• Intermediate filaments:
  • made up of a variety of proteins that share a common structure.
  • assemble into fibers that resemble a cable made up of twisted strands.
  • mechanical strength makes them ideally suited to provide structural support.
  • nuclear envelope has a network of intermediate filaments called the nuclear lamina.
• Microfilaments:
  • composed of the protein actin.
  • underlie the plasma membrane of animal cells, give them their characteristic
    shapes.
  • remodeling changes the shape of the cell.
  • important for cell movement.
  • play a role in cytokinesis.
• Microtubules:
  • made up of various kinds of a protein called tubulin.
  • play vital roles in cell division.
  • spindle fibers that attach to chromosomes during metaphase are made up of
    microtubules.
  • serve as tracks along which motor proteins (dynein and kinesin) transport
    cargo to different parts of the cell.
• Additional functions:
  • organizing the contents of the cell.
  • organelles anchored by attachment to the cytoskeleton.
  • interactions among cytoskeletal proteins and components of the extracellular
    matrix are crucial in maintaining tissue structure.
  • membrane-associated signaling proteins linked to components of the
    cytoskeleton; give cytoskeletal proteins a role in cell signaling pathways.

Tissues

• Cells in multicellular organisms are organized into tissues that play
  specialized roles in the body.
• Animals have four types of tissues in their bodies:
  • epithelium.
  • connective tissue.
  • nerve tissue.
  • muscle tissue.
• Epithelial tissues line the cavities and surfaces of blood vessels and organs.
  • Epithelial cells categorized by shapes: squamous, columnar, cuboidal.
  • Can be organized in a single cell layer or in layers (stratified or layered).
  • Glands are comprised of epithelial cells.
  • Cell functions include: protection, secretion, selective absorption,
    transport, and sensing.
  • Layers of epithelial cells do not contain blood vessels and must receive
    nutrients through diffusion from underlying connective tissue, through the
    basement membrane.
• Connective tissue:
  • serves as the “glue” to hold everything together.
  • fills the gaps between all the other tissues of the body, including the
    nervous system.
  • meninges (cover of brain) and spinal cord are composed of connective tissue.
  • Contains:
    • 1) cells.
    • 2) ground substance.
    • 3) fibers.

Introduction: Basic Chemistry

• Quote by Michael Adams: “Organic chemistry is the chemistry of carbon compounds.
  Biochemistry is the chemistry of carbon compounds that crawl.”
• To understand biochemistry, one must possess at least a basic understanding
  of organic and general chemistry.
• Chemistry is chemistry, whether in a cell or outside it, but biological
  chemistry is a particular subset of organic chemistry that often involves
  enormous macromolecules, and that happens in the aqueous environment of the
  cell. Figure 1.18 shows the various organic functional groups common in
  biochemistry.
• Students need a basic understanding of covalent and ionic bonds.

Covalent bonds

• Covalent bonds are the result of sharing of electrons between two atoms.
• Ionic bonds, by contrast, are formed when one atom donates an electron to another, such as in the formation of sodium chloride.

Electronegativity

• Electronegativity is a measure of the affinity a nucleus has for outer shell electrons.

• High electronegativity corresponds to high affinity Electronegativity is a measure of the affinity a nucleus has for outer shell electrons (Table 1.2). High electronegativity cor- responds to high affinity. Electrons in a covalent bond are held closer to the nucleus with a greater electronegativity compared to a nucleus with lower electronega- tivity.

• For example, in a molecule of water, with hydrogen covalently bonded to oxygen, the electrons are “pulled” toward the oxygen, which is more electronegative. Because of this, there is a slightly greater negative charge near the oxygen atom of water, compared to the hydrogen (which, correspondingly has a slightly higher positive charge). This unequal charge distribu- tion sets up a dipole, with one side being some- what negative and the other somewhat posi- tive. Because of this, the molecule is de- scribed as polar.

• Hydrogen bonds between water molecules are the result of the attraction of the partial positive and partial negative charges on differ- ent water molecules (Figure 1.20). Hydro- gen bonds can also form between hydrogens with a partial positive charge and other strongly electronegative atoms, like nitrogen, with a partial negative charge. It is important to remember that hydrogen bonds are interactions between molecules (or parts of molecules) and are not bonds between atoms, like cova- lent or ionic bonds.

• Bonds between hydrogen and carbon do not form significant partial charges because the electronegativities of the two atoms are similar. Consequently, molecules containing many carbon-hydrogen bonds will not form hydrogen bonds and therefore, do not mix well with water. Such molecules are called hy- drophobic. Other compounds with the abil- ity to make hydrogen bonds are polar and can dissolve in water. They are called hydro- philic. Molecules possessing both character- istics are called amphiphilic.

Weak interactions

• Hydrogen bonds are one kind of electrostatic (i.e., based on charge) interaction between di- poles. Other forms of electrostatic interac- tions that are important in biochemistry in- clude weak interactions between a polar molecule and a transient dipole, or between two temporary dipoles. These temporary di- poles result from the movement of electrons in a molecule. As electrons move around, the place where they are, at a given time, becomes temporarily more negatively charged and could now attract a temporary positive charge on another molecule. Since electrons don’t stay put, these dipoles are very short-lived.
• Thus, the attraction that depends on these di- poles fluctuates and is very weak. Weak interactions like these are some- times called van der Waals forces. Many molecular interac- tions in cells depend on weak inter- actions. Although the individual hy- drogen bonds or other dipole-dipole interac- tions are weak, because of their large num- bers, they can result in quite strong interac- tions between molecules.

Oxidation/reduction

• Oxidation involves loss of electrons and reduction results in gain of electrons. For every biological oxidation, there is a corre- sponding reduction - one molecule loses electrons to another molecule. Oxidation reactions tend to release energy and are a source of bioenergy for chemotrophic cells.

Ionization

• Ionization of biomolecules, by contrast does not involve oxidation/reduction. In ionization, a hydrogen ion (H+) leaves be- hind its electron as it exits (leaving behind a negative charge) or joins a group (adding a positive charge). Biological ionizations typi- cally involve carboxyl groups or amines, though phosphates or sulfates can also be ionized. A carboxyl group can have two ioni- zation states - a charge of -1 corresponds to the carboxyl without its proton and a charge of zero corresponds to the charge of the car- boxyl with its proton on. An amine also has two ionization states. A charge of zero corre- sponds to a nitrogen with three covalent bonds (usually in the form of C-NH2) and a charge of +1 corresponds to a nitrogen mak- ing four covalent bonds (usually X-NH3+).

Stereochemistry

• A carbon has the ability to make four single bonds (forming a tetrahedral structure) and if it bonds to four different chemical groups, their atoms can be arranged around the carbon in two different ways, giving rise to stereochemical “handedness” (Figure 1.21). Each carbon with such a property is re- ferred to as an asymmetric center. The property of handedness only occurs when a carbon has four different groups bonded to it.
• Enzymes have very specific 3-D structures, so for biological molecules that can exist in dif- ferent stereoisomeric forms, an enzyme that synthesizes it would make only one of the pos- sible isomers. By contrast, the same mole- cules made chemically (not using enzymes) end up with equal amounts of both isomers, called a racemic mix.

Gibbs Free Energy

• Gibbs Free Energy: The Gibbs free energy calculation allows us to determine whether a reaction will be spon- taneous, by taking into consideration two fac- tors, change in enthalpy ($\Delta H$) and change in entropy ($\Delta S$).

• Equation:

  • $G = H - TS$
  • $\Delta G = \Delta H - T\Delta S$
  • Note: A ΔG that is negative corresponds to release of free energy. Reactions that release energy are exergonic, whereas those that absorb en- ergy are called endergonic.

• Equilibrium constant:

  • The biological standard Gibbs free energy change ($\Delta G°’$) corresponds to the ΔG for a process under standard con- ditions of temperature, pressure, and at pH = 7.
  • For a reaction
    • $aA + bB <=> cC + dD$
  • the equilibrium constant, Keq is
    • $Keq = {([C]^c<em>{eq} [D]^d</em>{eq}) \over ([A]^a<em>{eq} [B]^b</em>{eq}) }$
    • where a,b,c, and d are integers in the bal- anced equation.
    • Note: Large values of Keq cor- respond to favorable reactions (more C and D produced than A and B) and small values of Keq mean the opposite.
  • At equilibrium
    • $\Delta G°’ = -RTlnKeq$

• Linked processes:

  • If a process has a $\Delta G= Z$ and a second process has a $\Delta G= Y$, then if the two processes are linked, $\Delta G$ and $\Delta G°’$ values for the overall reac- tion will be the sum of the individual $\Delta G$ and $\Delta G°’$ values.
  • $\Delta G<em>{total} = \Delta G</em>1 + \Delta G_2 = Z + Y$
  • $\Delta G°’<em>{total} = \Delta G</em>1°’ + \Delta G_2 °’$

Catalysis

• Catalysis is an increase in the rate of a reaction induced by a substance that is, itself, unchanged by the reaction. Because catalysts re- main unchanged at the end of a reaction, a sin- gle catalyst molecule can be reused for many reaction cycles.
• Proteins that catalyze reac- tions in cells are called enzymes, while ribo- zymes are RNA molecules that act as cata- lysts.

Introduction: Water and Buffers

• When it comes to water, we’re literally drown- ing in it, as water is by far the most abun- dant component of every cell.
• To understand life, we begin the dis- cussion with the basics of water, because everything that happens in cells, even reactions buried deep inside enzymes, away from water, is influ- enced by water’s chemistry.
• The water molecule has wide ‘V’ shape (the H- O-H angle is 104°) with uneven sharing of elec- trons between the oxygen and the hydrogen atoms (Figure 1.23). Oxygen, with its higher electronegativity, holds electrons closer to itself than the hydrogens do. The hy- drogens, as a result, are described as having a partial positive charge (typically des- ignated as δ+) and the oxygen has a partial negative charge (written as δ-). Thus, water is a polar molecule because charges are distrib- uted around it unevenly, not symmetrically.

Water as a solvent

• Water is described as a solvent because of its ability to solvate (dissolve) many, but not all, molecules.
• Molecules that are ionic or polar dissolve readily in water, but non-polar substances dissolve poorly in water, if at all. Oil, for example, which is non-polar, separates from water when mixed with it. On the other hand, sodium chloride, which ionizes, and ethanol, which is polar, are able to form hydrogen bonds, so both dissolve in water.
• Solubility in water is crucial for brewers, winemakers, and distillers – but for this property, there would be no wine, beer or spirits.
• Hydrophilic is substances that interact well with water and dissolve in it and hydrophobic to refer to materials that are non-polar and do not dissolve in water.
• Amphiphilic refers to compounds that have both properties. Soaps, for example are amphiphilic, containing a long, non-polar aliphatic tail and a head that ionizes.

Solubility

• The solubility of materials in water is based in free energy changes, as measured by ΔG. Re- member that H is the en- thalpy (heat at constant pressure) and S is entropy. Given this
  • $\Delta G = \Delta H - T\Delta S$
  • where T is the temperature in Kelvin. For a process to be favorable, the ΔG for it must be less than zero. From the equation, lowered ΔG values will be favored with decreases in enthalpy and/or increases in entropy.

Water organization

• Since non-polar molecules don’t really interact with water, the water be- haves very differ- ently than it does with ions or mole- cules that form hy- drogen bonds. In fact, around each non-polar molecule, water gets very or- ganized, aligning it- self regularly. As any freshman chemistry student probably re- members, entropy is a measure of disorder, so when something becomes ordered, entropy decreases, meaning the ΔS is negative, so the TΔS term in the equation is positive (negative of a negative).
• Since mixing a non-polar substance with wa- ter doesn’t generally have any significant heat component, the ΔG is positive. This means, then, that dissolving a non-polar compound in water is not favorable and does not occur to any significant extent. Further, when the non-polar material associates with itself and not water, then the water molecules are free to mix, without being ordered, resulting in an increase of entropy. Entropy therefore drives the separation of non-polar substances from aqueous solu- tions.

Amphiphilic substances

• We consider mixing of an amphiphilic substance, such as a soap, with water (Figure 1.24). The sodium ions attached to the fatty acids in soap readily come off in aqueous solution, leaving behind a negatively charged molecule at one end and a non-polar region at the other end.
• The ionization of the soap causes in an increase in entropy - two particles instead of one. The non-polar portion of the negatively charged soap ion is problematic - if exposed to water, it will cause water to organize and result in a decrease of entropy and a positive ΔG. Just like the non-polar molecules in the first example associated with each other and not water, so too do the non-polar portions of the soap ions associate with each other and exclude water. The result is that the soap ions arrange themselves as micelles with the non-polar portions on the interior of the structure away from water and the polar portions on the outside interacting with water.
• Interaction of the polar heads with water returns the water to its more disordered state. This increase in disorder, or entropy, drives the forma- tion of micelles. As will be seen in the discussion of the lipid bi-layer, the same forces drive glycerophospholipids and sphingolipids to spontane- ously form bilayers where the non-polar portions of the mole- cules interact with each other to exclude water and the polar portions arrange themselves on the outsides of the bilayer.

Yet another example is seen in the folding of globular proteins in the cytoplasm. Non- polar amino acids are found in the interior portion of the protein (water excluded). In- teraction of the non-polar amino acids turns out to be a driving force for the folding of proteins as they are being made in an aque- ous solution.

Hydrogen bonds

• The importance of hydrogen bonds in bio- chemistry (Figure 1.30) is hard to over- state. Linus Pauling himself said, “ . . . . I believe that as the methods of struc- tural chemistry are further applied to physiological problems it will be found that the significance of the hydrogen bond for physiology is greater than that of any other single structural feature.”